Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

Abstract

Pancreatic islets are highly vascularized and arranged so that regions containing beta-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic ( approximately 300 mg/dl or 16.8 mM) and hypoglycemic ( approximately 50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the beta-cells or yellow fluorescent protein in the alpha-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of beta-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

Models used for identifying and imaging islets in vivo. Islets within a living mouse with β-cells expressing enhanced green fluorescent protein (eGFP) (MIP-GFP; A, top) or α-cells expressing enhanced yellow fluorescent protein (eYFP) (Gluc-Cre-YFP; A, bottom) are used to locate these cells in relation to their neighboring vessels. Three-dimensionally resolved optical slices (Z-stacks) through the islet of a MIP-GFP mouse (B) and a Gluc-Cre-YFP mouse (C) show the peripheral location of glucagon cells in the mouse islet. D: single-plane confocal microscope image of an islet with GFP-labeled β-cells (green) and islet vasculature visualized with a Cy5.5 dextran tracer (purple). E: single-plane image of an islet in a mouse injected with sulforhodamine-labeled red blood cells.

Glucose clamp experiments. A: time line for experimental design shows that, after the mouse was anesthetized, the pancreas exteriorized, and islet located, blood glucose levels were measured every 10 min. For experimental design 1, where experiments were conducted first under hyperglycemia and then hypoglycemia, the mouse was given insulin continuously and varying amounts of glucose until the hyperglycemic state was achieved. A series of images was then taken to capture injection of fluorescent dextran as it filled the islet in the hyperglycemic state. The glucose infusion was terminated while insulin infusion was kept constant to achieve hypoglycemia (∼50 mg/dl). A second injection of fluorescent dextran was imaged in the hypoglycemic state. For experimental design 2, where experiments were conducted first under hypoglycemia and then hyperglycemia, the mouse was infused continuously with insulin until hypoglycemia was achieved, at which time images were taken. Following this, the mouse was infused with both insulin and glucose to achieve hyperglycemia, when another set of images was taken to capture the filling of the islet after a fluorescent dextran injection. B: glucose levels during a representative design 1 experiment where hyperglycemia was achieved before hypoglycemia. C: glucose levels during a representative design 2 experiment where hypoglycemia was achieved before hyperglycemia.

Islet blood flow during hyperglycemia and hypoglycemia. Images taken at peak fluorescence intensity in the same islet under experimental design 1 during hyperglycemia (A) and hypoglycemia (B) or under experimental design 2 during hypoglycemia (C) and hyperglycemia (D).

Vessel filling rate during hyperglycemia and hypoglycemia. A series of images taken from 0–2.5 s captures filling rate of islet vasculature during hypoglycemia (A–D) or hyperglycemia (E–H). By 1.5 s, the islet has completely filled under hyperglycemic conditions, whereas the islet is only partially filled at the same time point during hypoglycemia. No. of islets that exhibit faster filling rates in hyperglycemia vs. hypoglycemia using experimental design 2 (I) or experimental design 1 (J).

Regional differences in flow in hyperglycemia and hypoglycemia. Islet vasculature filled with fluorescent dextran at peak fluorescence intensity during hypoglycemia (A) or hyperglycemia (B). Arrows point to vessels that do not appear to be active in hypoglycemia (A) but are filled with fluorescent tracer under hyperglycemic conditions (B).